Immunofluorescence staining
This video explains chemical and antibody staining of cells and how to execute the experimental steps.Visualization of proteins is accomplished by antibody staining.
Description
Non-interactive video download link (mp4). Not available, view on YouTube instead.
The script that has been used to create the video shown above can be downloaded as excel script file. File not available.
| 1 | Check your calculation for diluting 1st AB with one of the assistants Dilute the 1st AB stock 1:2000 in blocking solution You will get the diluted 1st AB from the assistent |
| 2 | Aspirate blocking solution from the wells. |
| 3 | Add 100 µl 1st AB solution per well. |
| 4 | Wrap the plate in parafilm. Check the movie between step 2 and 3 of the interactive protocol, how to wrap parafilm around your plate. You just need a thin strip. |
| 5 | Incubate the plate overnight at 4°C. |
Above you can see your plate layout. Calculate the volume needed of the first antibody stock and the volume of blocking solution. You have plated 6 wells per conditions, however you will only stain 2 wells per conditions (n = 2). The dilution factor is 1:2000 and each well can hold 100 µL of liquid. On top of the amount that you need to fill all wells, add 2 wells of volume to your calculation to compensate for pipetting errors.
We need µL of the first antibody (minval=0.9, maxval=0.9)
We need µL of the blocking solution (minval=1790, maxval=1810)
When storing your samples/compound in a lab, it is important to write down the information of your sample on the package. What information should be written down on your first antibody solution tube:
Choose one or more options:
- (Team) name (correct)
- Date (correct)
- Dilution factor (correct)
- Second antibody name
- First antibody name (correct)
- Diluted in.. (correct)
- Tube size
All cells exhibit F-actin filaments. Why is it then useful to label F-actin?
Pick one
- F-actin forms a part of the cytoskeleton of the cell. The cell uses this to move, so that cell movement can be visualized.
- F-actin forms a part of the cytoskeleton of the cell. As this determines the cell shape it makes visualization of the individual cells possible. (correct)
- All cells exhibit F-actin filaments, but when the cell dies these filaments are broken down. The cell cannot be visualized then anymore; so, it is a viability marker.
- All cells exhibit F-actin filaments, but when the cell divides these change structure which can then be observed during imaging; so, it is a proliferation marker.
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- Skill levelIntroduction video
- CategoryCytochemistry
